DEAD-box RNA helicases are a versatile class of conserved proteins characterized by their ability to unwind RNA duplexes, which are believed to play important roles in regulating RNA function. However, many DEAD- box proteins have been poorly studied and hence their cellular functions are unknown. Without this knowledge, a detailed understanding of RNA metabolism remains incomplete. We propose to study two E. coli DEAD-box RNA helicases, SrmB and DeaD, which have been implicated in different aspects of RNA metabolism. Our preliminary data indicate the following: (i) SrmB and DeaD each promote a non-processive exo-ribonuclease, polynucleotide phosphorylase (PNPase), to digest structured RNA in vivo;(ii) the absence of either factor results in differential regulation of 8% of E. coli transcripts, presumably through an effect on RNA conformation and turnover;(iii) the cold-sensitive growth phenotype of srmB and deaD strains can be suppressed by over-expressing ribosomal RNA processing and modification factors;(iv) E. coli DEAD-box helicases stimulate the function of poly(A) polymerase (PAP), an enzyme that polyadenylates cellular transcripts and targets them for degradation. Therefore, each of these proteins has multiple roles in RNA turnover. Using a combination of genetic and biochemical approaches, the focus of the proposed studies will be to define these functions in detail. First, we will conduct a genome-wide survey to identify cellular transcripts that undergo turnover via SrmB or DeaD stimulation of PNPase function. We will also biochemically characterize the RNA determinants that are responsible for these properties. Second, we will identify transcripts that directly interact with SrmB or DeaD in the cell, and investigate the mechanism of differential transcript regulation by these factors. Third, we will investigate the basis for suppression of the srmB and deaD cold-sensitive growth defect due to the over-expression of ribosomal RNA processing factors. Fourth, we will analyze the mechanism and consequences of PAP stimulation by DEAD-box helicases. Overall, these studies should provide insights into the basis of target selection, functional interactions with RNA processing enzymes and the cellular functions of two important DEAD-box proteins. Such findings should be broadly relevant to DEAD-box proteins that have yet to be characterized in detail. PROJECT NARRATIVE By providing a greater understanding of RNA metabolism, the proposed studies on DEAD-box RNA helicases will provide better insights into diseases that are due to defects in RNA function. Advanced knowledge of such RNA helicases could also be helpful to design novel antibiotics that target conserved DEAD-box domains in pathogenic organisms.